Metformin exposure, maternal PCOS status and fetal venous liver circulation: A randomized, placebo-controlled study

Background Metformin is prescribed to women with polycystic ovary syndrome (PCOS) to prevent pregnancy complications. Children exposed to metformin vs. placebo in utero, have increased head circumference at birth and are more overweight and obese at 8 years of age. Also, maternal PCOS-status seems to alter the long-term cardio-metabolic health of offspring. We hypothesized that the long-term effects of metformin-exposure and/or maternal PCOS may be mediated by circulatory adaptations during fetal life. Material and methods This is a sub-study of a larger double-blinded, placebo-controlled trial, where women with PCOS were randomized to metformin (2g/day) or placebo in pregnancy, a total of 487 women. A sub-group of participants (N = 58) took part in this sub-study and had an extended ultrasound examination at gestational week 32, including blood flow velocity and diameter measurements of the umbilical vein (UV), the ductus venosus (DV) and the portal vein (PV). Blood flow volume was calculated and adjusted for estimated fetal weight (EFW) (normalized flow). Metformin exposed fetuses were compared to placebo exposed fetuses. Fetuses of mothers with PCOS (metformin [n = 30] and placebo [n = 28]) were compared to a low-risk reference population (N = 160) by z-score statistics. Results There was no difference in fetal liver flow between metformin vs. placebo-exposed fetuses. Fetuses of mothers with PCOS had higher EFW (0.63 [95% CI 0.44–0.83] p<0.001), lower normalized UV, DV, PV, and lower total venous liver blood flows than the reference population. Conclusion Metformin during pregnancy did not affect fetal liver blood-flow. In our population, maternal PCOS-status was associated with reduced total venous liver blood-flow, which may explain altered growth and metabolism later in life.


PCOS
The polycystic ovary syndrome (PCOS) affects some 8 -11% of women in the reproductive age group diagnosed according to National Institute of Health criter ia (NIH) (3)(4)(5) and probably up to 15-20 % according to The Rotterdam Consensus criteria.
The cornerstones of PCOS are insulin resistance and hyperandrogenicity, although none is mandatory for the diagnosis according to the Rotterdam criteria (6). PCOS has implications both for fertility and pregnancy outcome, while an increasing body of evidence points to a high prevalence of pregnancy complications (7)(8)(9)(10)(11). Hyperinsulinemia and hyperandrogenism have been suggested as pathogenic factors in both PCOS and pregnancy complications (12)(13)(14) .
Protocol for The PregMet 2 Study; version 7 2013-11-25 7 Preterm birth Data on women who had experienced preterm delivery in the county of Namdalen (Namsos hospital) shows a PCOS prevalence of 30 % (ref). The PCOS prevalence among controls who had given birth at term after an uncomplicated pregnancy was 15%.

The Pilot study
Our research group performed a pilot study on metfomin treatment of pregnant PCOS women in 2000 -2003. In this pilot study 7 out of 22 placebo-treated PCOS women experienced serious pregnancy complications, while none of the 18 metformin-treated women had such complications. These were promising results.

The PregMet 1 Study
To confirm these results, a Norwegian multicentre trial, the PregMet 1 Study, was conducted by Eszter Vanky as the principal investigator. However, metformin treatment from the first trimester to delivery, did not influence the incidence of gestational diabetes mellitus (GDM), Protocol for The PregMet 2 Study; version 7 2013-11-25 8 preeclampsia (PE), preterm birth (gestational week [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37], or the composite of these three end points when analysing the results according to "intention to treat principal" (2). "There is no study of metformin during pregnancy in women with PCOS that even comes close to this one in terms of scope and quality. This is today the alpha and the omega of metformin use in pregnancy in women with PCOS, the best level I evidence we have to counsel our patients…." (22) Preterm birth The WHO defines preterm labour as labour occurring before 37 weeks of pregnancy or before 259 days of pregnancy (A). As prognosis depends on gestational age at birth, we further subdivide preterm deliveries into following groups (23) 9 About 10% of preterm deliveries are extremely preterm, another 10 % very preterm and approximately 80 % moderately or late preterm deliveries.
The obstetric precursors leading to preterm birth are: 1. Delivery for maternal or fetal indications, in which labor is either induced or the infant is delivered by prelabor caesarean section; 2. Spontaneous preterm labor with intact membranes; and 3. Preterm premature rupture of the membranes (PPROM), irrespective of whether delivery is vaginal or by caesarean section (24). Preterm delivery is a major contributor to neonatal morbidity and mortality (27).
Epidemiological risk factors such as socioeconomic status and ethnicity seems to be important (26). Infections, uterine distension and anomaly are other known factors related to Protocol for The PregMet 2 Study; version 7 2013-11-25 10 preterm birth. However, the causes of preterm delivery are still essentially unknown and seem to be multifactorial (25).
Former preterm delivery is a significant risk factor. However, almost 50 % of preterm births occur without any identified cause or risk factor (25). The incidence of preterm birth is rising in most western countries, Norway included (28). This occurs despite all attempts to treat and prevent preterm birth.
The fact that former preterm labour is a strong risk factor, indicate a genetic or otherwise individual disposition. And the more or less universal rise in preterm birth rates in the Western world indicates that environmental or lifestyle risk factor changes are involved Incidence of preterm labour: Incidence in Norway (B) : 1980: 5.6 % 1995: 7.5 % 1999: 7.9 % Incidence in the USA (C): 1980: 8 % 1995: 12 % Although most preterm babies survive, they are at increased risk of neuro-developmental impairments, respiratory and gastrointestinal complications. Preterm infants are also of increased risk of developing cognitive and behavioral abnormalities and of achieving poorly in educational situations. These problems tend to last until adulthood (29). Recent evidence also indicate that infants born close to term (gestational week 34-36) carry a risk of increased morbidity and mortality risk, compared with term infants(E).
There are major socioeconomic costs connected to these patients. There are obvious costs due to initial neonatal treatment. But even more substantial is the health service costs following Protocol for The PregMet 2 Study; version 7 2013-11-25 11 discharge from the neonatal unit. This also represents long-term burdens, on affected families, but also on health, education and social services (30).
Recently published data on women who had at least one preterm birth, showed a PCOS prevalence of 25%, while 14% had PCOS among a control group of women who had given birth at term after an uncomplicated pregnancy (31). These data support former reports on preterm deliveries occurring more often among PCOS women and points to the fact that there is an increased prevalence of PCOS among women who experienced preterm birth.

Pooled data and per protocol analyses
In the PregMet 1 Study we found that, according to "intention to treat" analysis, 5 out of 135 metformin-treated and 11 out of 135 placebo-treated PCOS women experienced preterm birth (p=0.12). However, • Biologically and medically, late miscarriage (gestational week 13-21) and preterm birth is a "continuum", not separate conditions. Adding the miscarriage and preterm data in the PregMet 1 Study and analyzing it according to "intention to treat" principle, we find 13 late miscarriages/preterm births in the placebo group (N=135) and 5 in the metformin group (N=135), (p=0.055).

Aim of the PregMet 2 study
The overall aim and primary end point of the PregMet 2 Study is to investigate whether metformin prevents late miscarriages and preterm deliveries in PCOS women treated with metformin from first trimester of pregnancy to delivery in a large, randomized, controlled, multi-centre trial setting.

Oligo-amenorrea
Defined as a cycle length of ≥ 35 days or < 10 per year.

Hyperandrogenism
• Biochemical hyperandrogenemia: (s-testosterone ≥ reference value for women at each study site for the time being and/or s-SHBG ≤ 30 nmol/L).

Polycystic ovaries by ultrasound
Defined as at least 12 follicles in one ovary and/or an ovarian volume ≥ 10 ml (volume = 0.52 · d¹ · d² · d³) in at least one ovary. If there is a follicle ≥ 10 mm, repeat ultrasound at the time of ovarian quiescence Polycystic ovary (PCO) 12  Gestational age: Protocol for The PregMet 2 Study; version 7 2013-11-25 18 Gestational age is determined in the first trimester by a vaginal ultrasound examination.

Term date:
Term date is determined by a mid-second trimester, trans-abdominal ultrasound examination measuring BPD, mean abdominal diameter (MAD) and length of femur, or using the appropriate method that is standard at the actual study site.

Preterm birth:
Spontaneous or operative birth occurring before gestational week 37 + 0.

Hypertension in pregnancy:
Blood pressure measured after at least 10 minutes of comfortable chair rest ≥140/90 mmHg at two different occasions without proteinuria or other signs of preeclampsia occurring after week 20 in pregnancy.

Preeclampsia:
Protocol for The PregMet 2 Study; version 7 2013-11-25 19 Blood pressure measured after at least 10 minutes of comfortable chair rest ≥140/90 mm Hg at two different occasions and proteinuria (1+ protein in two different occasions or 2+ protein in one occasion on a urinary dip stick).

Gestational diabetes mellitus:
Gestational diabetes mellitus (GDM) is defined as a fasting plasma glucose ≥7.0 mmol/L or a 2 hour plasma glucose ≥ 7.8 mmol/L during a standard 75 g per oral glucose tolerance test (OGTT) performed according to the WHO standard.

Hirsutism:
Ferriman -Gallwey score ≥6 The endpoints of the study

Primary endpoint
The combined incidence of late miscarriages and preterm births.

Design
Prospective, randomized, double-blind, Nordic multi-centre study

Participating centres
Public or private, secondary or tertiary clinics dealing with gynaecology and obstetrics. We will invite centres form the Nordic countries. All principal investigators in all study centres have to be specialists in obstetrics/gynecology or under on-going specialist-training. • Gestational week at inclusion: week 6 + 0 to 12 + 6.

Inclusion criteria
• Wash out for metformin: at least 7 days • Able to communicate fluently in the official language at the study cite or English Protocol for The PregMet 2 Study; version 7 2013-11-25 22 • Informed consent
• Acute conditions that may worsen renal function, such as dehydration, septicaemia, cardiovascular collapse (shock), and intravascular administration of iodinated contrast.
• Acute or chronic conditions that can lead to tissue hypoxia, such as cardiac or respiratory failure, recent myocardial infarction or shock.
• Known hypersensitivity to any component of metformin

Study medication
• Metformin 500 mg x 2 daily the first week and then 1000 mg x 2 daily from the second week to delivery.
Protocol for The PregMet 2 Study; version 7 2013-11-25 23 • Placebo 1 tablet x 2 daily the first week. And then 2 tablets x 2 daily from the second week to delivery.
• To counteract a possible metformin action on folic acid and vitamin B12 levels, patients will be advised to take the daily dose of folic acid recommended in the country of the study centre. Further, one daily multivitamin tablet containing vitamin B6 and B12 will be recommended.
• Both metformin and placebo are produced by Weifa A/S. The tablets will be packed into boxes at the Hospital Pharmacy in Trondheim. After randomization the medication will be sent to cooperating pharmacies at the study site.
• The Hospital Pharmacy in Trondheim is responsible for medical accounting in the trial.
• A separate drugaccountibility log will be registered at each site.

Patients included in the PregMet 2 Study
• The overall intention and philosophy of the trial, is that included patients should receive the recommended and appropriate treatment available at each country and centre.
• Participation in the study does not limit any kind of diagnostic evaluation, examination or treatment option regarded as necessary during pregnancy.
• The participants in the PregMet 2 study can however not participate in other medical trials during the actual pregnancy.
Protocol for The PregMet 2 Study; version 7 2013-11-25 24 • Patients can only be included in the PregMet 2 Study once.

Progesterone treatment to prevent preterm delivery
In Iceland vaginal progesterone administration is used to prevent preterm delivery according to national guidelines; 1. In multiparous women who had former preterm delivery and 2. In women where cervix is found to be < 15 mm before gestational week 32. There are however no screening program for cervix length for asymptomatic women.
These treatment regimens interfere with the primary endpoint of the PregMet study. Therefore to avoid protocol violation and ethical conflict we decide the following: 1. At study centres in countries where progesterone is used prophylactic in multiparous women with former preterm birth, only nulliparous women will be included in the study.
2. In women with signs or symptoms of imminent preterm delivery, the routine treatment at the study centre should be given, -included vaginal progesterone treatment.
3. Vaginal progesterone treatment in such cases of imminent preterm delivery will be registered as a secondary endpoint.
If the progesterone treatment routine is introduced in other participating countries during the on-going study, these countries will adhere to the instructions above.
• Signed written informed consent.
Confirming the diagnosis of PCOS; anamnestically and from patient records

Blood samples
All blood samples will be collected in the morning between 08 and 11, and after an overnight fast. All screening analyses will be analysed at the local hospital.
Ten (10) test tubes of whole blood from each patient are centrifuged after 20 minutes, and serum is filled in nine (9) cryotubes with at least 1.8 ml of serum in each, and frozen at -70 º C (or -80 º C if that is preferred).Four EDTA tubes with whole blood will be filled, turned up and down a couple of times and transferred into cryotubes and freezed.
Two tubes for "buffy coat" will be separately centrifuged, processed and freezed.
Protocol for The PregMet 2 Study; version 7 2013-11-25 31 All cryotubes will be marked with a unique barcode. The tubes will be marked with a colour code for each visit. (Red hat for inclusion, yellow in week 19, blue in week 28, green in week 32, brown in week 36. Umbilical cord blood will be sampled at delivery (optional). We aim to have both venous whole blood samples, and both arterial and venous serum samples (Handled as described above). These will be marked with a purple hat.
All serum and whole blood will be sent to: Tone Shetelig Løvvik/ or study midwife,

Study duration
For each woman the participation in the present study will last from inclusion till 8 weeks post partum. The study medication will be stopped at delivery, but data will be collected and adverse events reported until the 8 th week post partum.

"Drop outs"
Data will primarily be analyzed according to the "intention to treat" principle, therefore data from patients who stop medication or do not present at pre-scheduled visits will also be included in the data analysis.
Protocol for The PregMet 2 Study; version 7 2013-11-25 32 Clarification: «Drop-out»: is a general term for those patients included in a study who • Clearly state that they do not want to continue in the study, (-they will not meet at pre-scheduled visits in the study) OR • Clearly state that they do not intend to take any more study medication OR • Clearly state that they do not want any contact with the study investigators NOT "Drop-out": patients who • Miss/forget an appointment, but attend other visits • Reduce or do not take study medication because of side effects Forget to take their study medication, but intend to take it.

In our "intention-to-treat analyses" we will include data from all "drop-outs". This is the meaning of "intention-to-treat".
Data included from drop-outs are: • already collected data and samples and • Information from delivery mother/baby. (The informed consent has not been withdrawn) • Already collected data will not be destroyed or deleted routinely, but if the patient explicitly asks for it (withdraws informed consent); we will delete it and not collect information from her delivery and baby at delivery.

Withdrawal from the study
Patients may withdraw from the trial at any time. If a patient withdraws from the trial she will be asked if the material already collected and information about the future delivery can be collected and included in the trial data base. If the patient requests, all information about the patient will be withdrawn. This is described in the informed consent. We wish to include and follow 1000 women in our trial. In our former studies withdrawal was a very minor problem, thus we expect a similar pattern in this trial, indicating no substitute is necessary. If this Protocol for The PregMet 2 Study; version 7 2013-11-25 33 proves to be a problem in accordance to keep our desired sample size, we might open up to include additional patients as long as the inclusion period is not stopped.

Adherence
Adherence will be evaluated by asking the participants about their tablet intake in a systematic manner at each visit. In addition participants will be asked to count and report their remaining tablets in connection with the telephone interview at 8 weeks postpartum. Further monitoring of the number of tablets is not required.
Good adherence is defined as consumption of ≥ 90% of the estimated drug dose during the study period. Acceptable compliance will be defined as drug consumption ≥ 70% and < 90%.
Poor compliance is defined as < 70% drug consumption.

Height
Will be measured in cm. Measured without shoes, onceat the inclusion visit

Weight
Weight will be measured at each prescheduled visit, without shoes and with light clothes on.

Weight in early pregnancy and weight development through pregnancy is an important factor
in almost all conditions in pregnancy.. Weight will be measured and approximated to the nearest kg.

Fasting plasma glucose and OGTT
The prevalence of gestational diabetes is high among PCOS. It is important to identify impaired glucose tolerance and GDM as early as possible in pregnancy and treat it appropriately

The Doppler examination of the uterine artery
Up to date ultrasound devices in daily practice will be used. Doppler ultrasound will be measured by abdominal approach. The point where the uterine artery crosses the internal iliac artery will be identified and the gate will be placed within 1 cm on each side of this point. The angle will be kept below 30 degrees. Pulsatility index (PI) will be measured on the right and left side 3 times. The lowest PI (LPI) for each side will be used in further analyses. Presence of a diastolic notch will be registered on each side.

Breast volume estimation
The volume of the breasts will be estimated by specially designed cups. This will be done at inclusion and at the last visit in pregnancy. See appendix.

Randomization
Randomization will be performed at the Unit for Applied Clinical Research (AKF),

Department of Cancer Research and Molecular Medicine, Norwegian University of Science
and Technology, Trondheim, Norway. Randomization will be in blocks of ten (5 metformin and 5 placebos) for each country. An unknown sized block will be introduced at the beginning of the inclusion in each country to make it impossible to estimate the actual study allocation of the participants.

Interim analyses
No interim analyses will be performed.

Blinding
The investigators at the study centers will enter patients into the web CRF at Unit for Applied Clinical Research. The participants then get a unique CRF number. The national pharmacy distributing the study medication will send the study medication to the including center. The study medication will be handed out by the investigator. When the participant is randomized, she will get a randomization number. The study drug package will have the same number as the patients randomization number.
The participants and care providers will be blinded for treatment allocation.

Unblinding
In a case of emergency unblinding or breaking the codes might be necessary. The sponsor/Dr. This information is found in the site file at each study center. .

Side effects
The most common side effects are gastrointestinal discomfort. Gastrointestinal side effects, headache and dizziness will be registered in a structured form in the CRF. Other possible and suspected side effects will be documented.
In case of suspected unacceptable side effects, the study medicine dosage will be adjusted or stopped.
All medication used by the participants will be registered.

Definition Adverse event (AE)
An adverse event (AE) is defined as any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product, which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavourable and Protocol for The PregMet 2 Study; version 7 2013-11-25 37 unintended sign (including an abnormal laboratory finding), symptom, or disease temporarily associated with the use of a medical product, whether or not related to the medical product.

Serious adverse events (SAE)
Any serious adverse events (SAE) arising during the study must be reported to the principal investigator of the study within 24 hours. SAE is defined as any untoward medical occurrence that at any dose: • Results in death, • Is life-threatening (i.e. the patient was at risk of death at the time of the event. It does not refer to an event, which hypothetically might have caused death if it was more severe).
• Requires in-patient hospitalisation or prolongation of existing hospitalisation.

SAE not to be reported;
Conditions to be reported as study endpoints, and not as SAE, even if they are leading to hospitalization.

Concomitant medication
Concomitant medication will be registered by generic name and treatment duration.
Medications taken for frequently occurring minor conditions in pregnancy (listed above) will not be registered.
The participants should not take any other vitamins, trace elements, dietary supplements or herbal medicine as long as they participate in the study. Fish oil and omega 3 can be taken

Data collection and management
Data collection and management will be performed by the internet based solution (web-CRF) at the Unit for Applied Clinical Research (AKF) in Trondheim. All women who sign the consent of participation and are screened for participation will be allocated a CRF number in an electronic clinical research form (CRF). Those who are randomized will also have a randomization number. The principal investigator in charge at each study centre keeps a code log with the CRF number allocated to each study participant. A copy of this code log will be kept by the Principal Investigator at each study site.
The web-CRF will be structured and will be completed at each consultation. Original patient files and results from blood tests will be filed for at least 15 years at each study site.
In the database (web-CRF) the patient will only be registered by a CRF number, initials (the first two letters of the given name and the first letter of the family name; ex: Anne Pettersen=ANP), age at inclusion and study site.

Management of the study
The study will be performed according to a "penta-blind" approach.
1. The patient will be blinded for the study medication.

2.
The investigator will be blinded for treatment allocation. This is the traditional "double-blind" design and represents the gold standard of pharmaceutical research; randomised double-blinded studies.

3.
Obstetricians who evaluate the end points (quality check the given diagnoses) of the study will not be among those who have consulted or treated of the participants during the study. To minimize the chance to estimate the treatment allocation of the individual patient, those who evaluate end points, will only be given access to the data they need to classify the outcomes, adverse events and safety data. When this is done and the monitoring is finished the data base will be locked.

4.
All statistical procedures will be performed by a statistician blinded for the actual medication; groups will be analysed only as group 1 and group 2.

5.
All results presented in tables and figures, which will be part of the first major publication from the PregMet 2 Study, will be worked out before the codes are broken.
This blinding procedure will be documented at the Unit for Applied Clinical Research (AKF) in Trondheim.

Sub-studies of the PregMet2 study
There are 3 sub-studies connected to the PregMet2 study 1. The RespPreg study "Metformin in lung health in pregnant women with PCOS" The sub-study has a questionnaire part (run in Bergen, Trondheim and Ålesund) and a spirometri test part (run in Bergen and Trondheim).
See separate protocol, CRF and patient information/consent.

The Polymeter study "Polycystic ovary syndrome metformin elimination research"
The sub-study collects extra blood samples in pregnancy and post partum. (The substudy is run in Trondheim) See separate protocol and patient information/consent. o Unit for Applied Clinical Research will support and administer some of the practical economic issues.

Statistical analyses
Statistical analysis of the data will be performed when all data are collected and quality checked. Data will be analyzed by an experienced statistician at Unit for Applied Clinical Research according to the "pentablind" approach.
Protocol for The PregMet 2 Study; version 7 2013-11-25 45 The main outcome (late abortions and preterm births) will be the end points by intention to treat (ITT) analyses. However, secondary "per protocol" analysis will also be performed. The metformin group will be compared to the placebo group using non-paired parametric and non-paired non-parametric methods as appropriate. Only two-sided tests will be used.

Risk of type I errors
The probability of a type I error is the probability of rejecting the null hypothesis when the null hypothesis is true. This is commonly referred to as the significance level of a test. Since we consider late miscarriages and preterm delivery to be a continuum, our study has only one primary endpoint. With only one primary endpoint, and a significance level of p = 0.05 and two-sided test we consider the risk of type I error extremely small and acceptable.

Modes of testing
After having conducted a virtually identical study on 273 pregnant women with PCOS, there is no reason to expect significantly more late miscarriages or preterm deliveries in the metformin group then in the placebo group. In spite of this we will only perform two-sided testing of our results.

Subgroup analysis
Subgroup analysis will be performed based on PCOS phenotype and pre-pregnant BMI below and above 30 kg/m 2 .

Power calculation
Based on the results from pilot and PregMet 1 Study the combined incidence of late miscarriages and preterm birth in women in the control (placebo) arm of the PregMet 2 Study will be at least 10%. Data from the PregMet 1 Study indicate a 60% reduction and data from the pilot study even more. We aim to show a 50% reduction with a power of 85% and alfa = 0.05. To do so, we need to include 500 patients in each treatment group.

Insurance
The Norwegian participants will be insured by the "Legemiddelansvarsforeningen".
Participants in other countries will be insured by a similar instance in each country.

Good Clinical Practice (GCP)
The study will be conducted in full compliance the GCP standards given in the guidelines.
The investigators are responsible for conducting the study in full accordance with the clinical study protocol, the latest version of the Declaration of Helsinki, and Good Clinical Practice: http://ec.europa.eu/health/files/eudralex/vol-10/3cc1aen_en.pdf

Monitoring
In Norway personnel from Unit for Applied Clinical research will monitor the study. In the other participating countries separate CRO organizations will be used for monitoring.
Monitoring will include: -All the informed consents -All SAE and SUSARs -The first three patients will be fully monitored at each study site.
-The next four patients; inclusion and exclusion criteria will be monitored and the inclusion visit, the visit in week 28 and the report from delivery. All in all approximately 20 % of the total data in each country will be monitored.

Publication
We aim to publish the results in international medical journals of the highest possible rank. In the main manuscript Tone Shetelig Løvvik is first author, Sven M. Carlsen is second author and Eszter Vanky is last author. Others are co-authors when they fulfil the Vancouver Declaration for authorship. The principal investigators in each country come after the second author, thereafter co-authors are included in an order that reflects the number of patients included at their study site.

Access to the material
Tone Shetelig Løvvik, Sven M. Carlsen and Eszter Vanky will have equal access and possibility to use the data and biological material collected in the study for further research beyond the main paper. When doing so all three will be given possibility to participate in the subprojects to a degree that justifies authorship on the particular sub-project.
Sven M. Carlen will be principal investigator and responsible for the possible long term follow up of the offspring during youth, adolescence and adulthood.
Tone Shetelig Løvvik, Sven M. Carlsen and Eszter Vanky will have equal access and possibility to use the data and biological material collected in the possible follow-up study/s of the offspring.
We will create a consortium study group consisting of the principal investigator from each participating country and Sven M. Carlsen and Eszter Vanky. The major focus of this consortium group should be to get the highest scientific value possible out of the biological material gathered during the PregMet 2 study.
If any of the investigators have an idea for a project, she/he can present a protocol and apply the consortium group for material from the study. Given the protocol has received an Protocol for The PregMet 2 Study; version 7 2013-11-25 51 ethical approval and there is no conflicting interest with the planned study and sub-studies the consortium group should as a general rule approve the study if it is sound and of superior scientific value. However, the consortium study group should always balance approval for projects against the fact that the long term interest for the biological material sampled in the PregMet 2 study will increase with time due to the uniqueness of the material and the possibility to follow up the mothers and children.

Ethics
Our calculations on the effect of metformin on late miscarriages and preterm deliveries based on a conservative estimate from former studies, indicates a potentially cheap, safe and effective prevention of preterm deliveries in PCOS women. Metformin is cheap and the patent protection period expired many years ago. This means that the pharmaceutical industry does not find it worthwhile conducting studies on possible metformin effects in pregnancy.
Based on data the NNT to prevent one preterm birth is around 10. However, based on our conservative power calculation the NNT is around 20 to prevent a preterm birth. If a positive effect of metformin on preterm deliveries can be proven in a large-scale multicentre study, it would be the most cost effective preventive treatment ever. Given, the short and long term benefit for the patients and the families involved and the potentially large economic savings for the society, it seems unethical not to perform the study.
Metformin has never been reported to be toxic or do any harm to the fetus or to the mother. This was also confirmed in the PregMet 1 study. Metformin do however have some gastrointestinal side effects that in some cases can be unpleasant.
Protocol for The PregMet 2 Study; version 7 2013- 11-25 52 In Norway or other Nordic countries, there is no specific follow up program for PCOS women in pregnancy. Given the extensive and structured follow up during pregnancy described in the present study, of a group of women with increased risk of pregnancy complications and suboptimal pregnancy outcome, we believe that participating in the PregMet 2 study will be beneficial also for the women and fetuses allocated to the placebo group.

Future plans
It is our intention to follow up both the women and their children. This includes: • Follow up the participants with a questionnaire 1 year after delivery about their general health and breast feeding experiences • To follow up the mothers long term to study the impact of GDM on long term glucose homeostasis.
• To follow up the offspring in early childhood and adolescence regarding endocrine and metabolic status.
• To merge data base, such as the Medical Birth Registry with data from the PregMet 2 study.
• Perform genetic analyses on whole blood and placenta, • Perform additional analyses on inflammatory markers and hormones.
For the follow up studies separate protocols and applications for regulatory authorities will be prepared.